Transmission electron microscopy is a technique widely used in the disciplines of biological and materials sciences. At its core, it relies on the ability to image through an object, following the interaction of electrons with the specimen. This allows the operator to observe a specimen at high spatial resolution for the purpose of identifying composition and structure in relation to a macroscopic property. An example in biological sciences is the study of the chemical synapse of the neuron, whereby transmission electron microscope images of the synapse aid the understanding of complex chemical processes in cell-to-cell signaling. An example in the materials science field would be using a transmission electron microscope to image an interface between two or more materials in an attempt to understand the relationship between the interfacial structure and some externally measured macroscopic physical property. In both examples, the ability to obtain images in three dimensions assists the user in determining the spatial relationship between discrete elements of an image, therefore providing a better understanding of complex interactions occurring at length scales ranging from micrometers (μm) to picometers (μm).
Specimen holders for the TEM are of two distinct types: top-loading and side-entry. In the case of a top-loading specimen holder, the TEM specimen is loaded vertically into the TEM via a complex transfer mechanism. The principal advantage of top-loading specimen holders is their lack of direct connection to the exterior surfaces of the TEM, resulting in less holder drift, which is an important consideration when obtaining high-resolution TEM images. However, the majority of microscopes that operate up to 400 keV utilize side-entry specimen holders since these offer significantly greater advantages. Generally, side-entry specimen holders are larger, therefore allowing more complex manipulation of the specimen, such as rotation and strain application, and increasing the ability to perform X-ray analysis (EDS).
The most basic of TEM holders for analyzing specimens is the single-tilt holder. Besides allowing movement in the Z plane for adjusting the height of the specimen to a eucentric position, basic specimen holders are limited to movement in the X or Y planes perpendicular to the electron beam and to plus/minus rotation in the longitudinal direction of the X axis. These holders are typically robust and reliable. They can give the user some information as to the behavior of the specimen under the electron beam. This would include changes in diffraction contrast with rotation about the X axis.
However, with the need for more advanced analytical TEM techniques, the information derived from a single-tilt holder is often too limited. This is particularly true when imaging or performing diffraction studies of crystalline materials. In this case, users would opt for a double-tilt holder in which the two tilt angles are perpendicular to each other. This allows tilting of a crystalline specimen to a degree that alignment with a particular crystallographic orientation is possible. Such holders utilize mechanical means to tilt the specimen in the Y axis as well as the X axis.
In certain cases, in addition to tilting, it is necessary to rotate the specimen. Two examples of this would be using electron tomography for three-dimensional reconstruction, and stereo pair imaging for the analysis of planar structures such as grain boundaries and interfaces. In both cases, the specimen is rotated until the features of interest align with the tilt axis of the specimen holder. Holders that are capable of rotating the specimen in combination with tilt along a single axis have been commercially available for some time. More recently, holders that can combine rotation with tilt along two axes have become available. These address the specific need for the stereological analysis of crystalline materials when, as the user tilts the specimen, the contrast changes, therefore complicating the analysis. To reduce this effect, the user can rotate and align the tilt axis to the specimen's g vector. Using this approach of rotating and tilting the specimen has a reduced impact on changes in diffraction contrast conditions.
There are two primary methods for obtaining three-dimensional images in a transmission electron microscope: stereo pairs and tomography. Stereo pairs are typically derived from tilting a specimen over a range of +/−10° to as much as +/−25°, with an image taken at both outermost angles. Applying the known tilt angle, the microscopist can then overlay these two images using either an analog or a digital technique so that the left and right eyes separately, rendering a three-dimensional image to the viewer.
In the case of tomography, a high-tilt tomography holder is used to tilt the specimen over a range of +/−70° to +/−80°. Images are obtained at predetermined angular increments, often as little as 1°, and then reconstructed digitally to give a three-dimensional representation of the specimen. Fiducial markers, often in the form of gold nanoparticles, are placed on the specimen and used as reference markers to assist in aligning each image within the tomogram. Besides biological applications such as the analysis of the neuronal synaptic junction, tomography is utilized by materials scientists for the three-dimensional reconstruction of the arrangement of atoms; for example, to study nanoscale materials, particles or interfaces. To collect tomograms at high magnification and with atomic resolution is particularly challenging. The field of view may be as small as 25×25 nm and features of interest such as vacancies or atoms in the region of 1 nm or less.
When trying to move the specimen over small distances, two major limitations in current tilt-and-rotate holders dominate. These are backlash and stiction. Backlash is uncontrolled movement associated with the inherent lack of precision in the mechanical drive mechanism, and stiction is static friction associated with mechanical contact between materials surfaces.
In current designs, there are no intrinsic mechanisms for digitally driving the specimen holder since there are no real-time feedback systems to identify the position or orientation of the holder. That is not to say that such a feedback mechanism could not be introduced into current holder design. Its lack of inclusion is more than likely associated with the current philosophy of simplicity and reliability of mechanical systems and the desire to minimize electromagnetic interference (EMI) near the specimen.
The advantages of introducing micromachined MEMS technology into current holder designs would include both digital control as well as the ability to move small distances reliably and routinely. The recent introduction of silicon-on-insulator (SOI) processes in MEMS devices has led to the development of inchworm drives that are capable of moving structures relatively large distances (several mm) and with high precision (40+nm per iterative step). However, this technology alone suffers from a general lack of sufficient applied force to move a beam or cantilever, and only with careful design is it possible to overcome stiction.
In addition to MEMS-based technology, this invention utilizes PZT technology, which can generate sufficient power to move MEMS-based structures and, depending on the applied voltage, can control movements in the 10-nanometer to several-micrometers range, ideal for controlled specimen tilt in TEM holders. The degree of tilt is governed by the design of the MEMS device and the applied voltage on the PZT. More specifically, this invention addresses the inability of commercially available specimen holders to allow rapid tilting forward and backward of the specimen within the electron beam to produce real-time stereo or tomographic images.
To the human eye, producing an image refreshed at 50 frames per second (i.e. every 20 milliseconds) would be interpreted as a real-time image to the brain. By integrating MEMS into the TEM specimen holder, the specimen can be tilted over a defined angle at up to 1000 cycles per second. By synchronizing the rapid dynamic tilt of the specimen with image collection on a CCD camera, real-time stereo images and tomograms can be obtained. This method of image collection can have a significant impact when dealing with electron beam-sensitive materials such as biological materials and polymers. In addition to collecting three-dimensional images, using this approach also makes it possible to collect a time series of images. This offers the user the opportunity to determine the relative beam sensitivity of components of an image through changes in structure or contrast as a function of time or electron dose. By following the change in the image as a function of time and/or dose, the user would be able to compute and reconstruct a zero dose image.